Purification of GIP / GLP-1 dual agonist peptides

Abstract

The present invention provides for purification of Tirzepatide using selective ion- pairing agents in the reversed phase-high performance liquid chromatography, for purifying crude Tirzepatide from closely related impurities.

Description

[0001] PURIFICATION OF GIP / GLP-1 DUAL AGONIST PEPTIDES

[0002] Related Application:

[0003] This application claims the benefit of priority of our Indian patent applications IN 202441046451 filed on June 17, 2024 which is incorporated herein by reference.

[0004] TECHNICAL FIELD OF THE INVENTION

[0005] The present invention relates to a method for purifying crude GIP / GLP-1 dual agonist peptide Tirzepatide which is represented by the Formula-!.

[0006] Formula-I

[0007] BACKGROUND AND PRIOR ART OF THE DISCLOSURE

[0008] Tirzepatide is a 39-amino-acid modified peptide based on the GIP sequence.

[0009] Tirzepatide contains 2 non-coded amino acids (aminoisobutyric acid, Aib) in positions 2 and 13, a

[0010] C -terminal amide, and Lys residue at position 20 that is attached to 1,20- eicosanedioic acid via a linker. The molecular weight is 4813.53 Da and the empirical formula is C225H348N48O68.

[0011] Tirzepatide (MOUNJAROTM) was developed by Eli Lilly Co and was initially approved in United States in 2022 as subcutaneous injection.

[0012] Several attempts for purification of GLP-1 analogues including Tirzepatide have been reported in the past. W02005019262 discloses the purification of Glucagon like peptide by reversed phase high performance liquid chromatographic process wherein the solvent used for elution is pH- buffered in the range from about pH 4 to about pH 10.

[0013] W02005019262 discloses purification of Glucagon like peptides using Tris buffer as mobile phase.

[0014] CN112661815B discloses the use of Acetonitrile as Mobile phase for purification of Tirzepatide.

[0015] SUMMARY OF THE INVENTION

[0016] Aspects of the present application provides processes for purification of Tirzepatide.

[0017] One aspect of the present invention provides for a method of purifying crude Tirzepatide by RP-HPLC method using cationic ion pairing agent.

[0018] Another aspect of the present invention provides for a method of purifying crude Tirzepatide, by RP-HPLC method using a cationic ion pairing agent to resolve closely associated impurities at RRT’s 0.90, 0.92, 0.96, 0.97, 0.98, 1.02, 1.05 & 1.06.

[0019] Yet another aspect of the present invention provides for a method for purifying crude Tirzepatide, the method comprising of a) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a cationic ion pairing agent along with the buffer b) subjecting the Tirzepatide from step a) to a second RP-HPLC purification using a anionic ion pairing agent along with the buffer c) Isolating the purified Tirzepatide.

[0020] Wherein, optionally steps a or b or steps a & b are repeated, and steps a & b are performed in reverse order. Another aspect of the present invention provides for a method of purifying crude Tirzepatide, the method comprising of: a) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a cationic ion pairing agent along with the buffer with basic pH b) subjecting the Tirzepatide from step a) to a second RP-HPLC purification using a anionic ion pairing agent along with the buffer with acidic pH c) Isolating the purified Tirzepatide.

[0021] Wherein, optionally steps a & b are repeated.

[0022] Another aspect of the present invention provides for a method of purifying crude Tirzepatide, the method comprising of: a) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a cationic ion pairing agent along with the buffer with basic pH b) subj ecting the Tirzepatide from step a) to a second RP-HPLC purification using a cationic ion pairing agent along with the buffer with basic pH c) subjecting the Tirzepatide from step b) to a third RP-HPLC purification using a anionic ion pairing agent along with the buffer with acidic pH d) Isolating the purified Tirzepatide.

[0023] Another aspect of the present invention provides for a method of purifying crude Tirzepatide, to resolve closely associated impurities atRRT’s 0.90, 0.92, 0.96, 0.97, 0.98, 1.02, 1.05 & 1.06.

[0024] Another aspect of the present invention provides for a method for purifying crude Tirzepatide, the method comprising of: a) dissolving the crude Tirzepatide using a cationic ion pairing agent along with the buffer b) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a cationic ion pairing agent along with the buffer c) subjecting the Tirzepatide from step a) to a second RP-HPLC purification using a anionic ion pairing agent along with the buffer d) Isolating the purified Tirzepatide.

[0025] Wherein, optionally steps a & b are repeated.

[0026] Another aspect of the present invention provides for a method for purifying crude Tirzepatide, the method comprising of: a) dissolving the crude Tirzepatide using a anionic ion pairing agent along with the buffer b) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a anionic ion pairing agent along with the buffer c) subjecting the Tirzepatide from step a) to a second RP-HPLC purification using a cationic ion pairing agent along with the buffer d) Isolating the purified Tirzepatide.

[0027] Wherein, optionally steps a & b are repeated.

[0028] Another aspect of the present invention provides for a method for purifying crude Tirzepatide, wherein the cationic pairing agent is selected from a quaternary ammonium compound and anionic pairing agent is selected from alkane sulfonic acid.

[0029] Another aspect of the present invention provides for a method for purifying crude Tirzepatide, wherein the alkane sulfonic acid is selected from the group consisting of octane sulfonic acid and hexane sulfonic acid.

[0030] Another aspect of the present invention provides for a method for purifying crude Tirzepatide, wherein the quaternary ammonium compound is selected from the group consisting of tetra butyl ammonium phosphate, tetra butyl ammonium chloride, decyl trimethyl ammonium, and tetra butyl ammonium hydroxide. Another aspect of the present invention provides for a method for purifying crude Tirzepatide,

[0031] Wherein basic aqueous buffer is selected from Tris-HCl, ammonium acetate, ammonium bicarbonate, Phosphate buffer, and citrate buffer.

[0032] Another aspect of the present invention provides for a method for purifying crude Tirzepatide,

[0033] Acidic aqueous buffer is selected from formic acid, Trifluoro acetic acid, phosphoric acid, perchloric acid and perchloro acetic acid.

[0034] Abbreviations:

[0035] GIP: Glucose-dependent insulinotropic polypeptide

[0036] GLP: Glucagon like peptide

[0037] RP HPLC: Reversed Phase High Performance Liquid Chromatography

[0038] HPLC: High Performance Liquid Chromatography

[0039] Fmoc: Fluorenylmethyloxycarbonyl tBu: Tertiary butyl

[0040] API: Active Pharmaceutical Ingredient

[0041] RRT : Relative Retention Time

[0042] OSA: Octane sulphonic acid

[0043] HSA: Hexane sulphonic acid

[0044] TBAP: Tetra butyl ammonium phosphate

[0045] TBA-CL: Tetra butyl ammonium chloride

[0046] DTMA: Decyl trimethylammonium

[0047] TBA-OH: Tetra butyl ammonium hydroxide

[0048] Tris-HCl: Tri s(hydroxymethyl)aminom ethane hydrochloride

[0049] ACN: Acetonitrile

[0050] IPA: Iso-Propyl Alcohol

[0051] NaOH: Sodium hydroxide

[0052] Tirze: Tirzepatide Advantages of present invention:

[0053] Peptide drug purification process development is a long and costly process, mostly involving different types of HPLC techniques, starting with the screening using different types of techniques, systems either as single HPLC or in combination with different types of HPLC steps. These steps are considered the cornerstone of drug purification, with solid-phase peptide synthesis with the Fmoc / tBu strategy being one of the most used. Therefore, truncations and deletions become significant sources of contamination, even with high coupling efficiencies.

[0054] The above prevalent problems have been overcome by the present invention which provides a robust, cost-effective and high yielding purification process of Tirzepatide.

[0055] Crude Tirzepatide (Assay -40%; Purity -20%) is subjected to sequential RP-HPLC purification steps under diverse conditions, followed by lyophilization to yield pure Tirzepatide. The present invention involves using selective ion-pairing agents in basic and acidic pH environments while purifying using reverse phase high performance liquid chromatographic (RP-HPLC) steps, for purifying crude Tirzepatide from closely related impurities. Absence of ion pairing agents in acidic and basic pH environments (mobile phases) in the process results in no or poor resolution of the related impurities, leading to retention of impurities in the final API. We surprisingly found through extensive investigations and experiments, that when first RP-HPLC purification’s is done under basic condition along with a cationic ion pairing agent followed by RP-HPLC using acidic conditions along with an anionic ion pairing agent resulted in higher purity with resolution of closely associated impurities and with greater yield compared to the vice-versa route.

[0056] The invention provides for the purification of very closely associated impurities. These closely associated impurities formed in the present process for preparation of Tirzepatide are present in RRT 0.89, 0.90, 0.94, 0.97, 1.02, 1.05, 1.06 & 1.07. Removal of these impurities with high throughput yield is the critical challenge and present invention has provided the solution for this problem. Summary of the advantages are as below, the invention provides for a) Selectively using particular type of ion pairing agent for selective removal of specific impurity at particular stage using RP-HPLC. b) Carrying out next stage RP-HPLC containing different type of ion pairing agent for selective removal of another type of specific impurity. c) Followed by pH treatment and isolation.

[0057] Comparison of Tirzepatide purity profiles without and with the anionic ionpairing agent octane sulfonic acid, and the cationic ion-pairing agent tetrabutylammonium phosphate.

[0058] The table below provides a comparison of Tirzepatide purity profile with respect to closely associated impurities present at RRT’s 0.90, 0.94, 0.97, & 1.02 without & with usage of Octane sulfonic acid as ion paring agent.

[0059] OSA helps in resolution of closely eluting impurities such as 0.94, 0.97 & 1.02 RRT which are critical for Tirzepatide purification.

[0060] The below table provides a comparison of Tirzepatide purity profile with respect to closely associated impurities present at RRT’s 0.90, 0.92, 0.94, 0.97, 0.98, 1.02, & 1.05 without & with usage of Tetra butyl ammonium phosphate as cationic ion paring agent.

[0061] TBAP helps in resolution of closely eluting impurities such as 0.98 & 1.02 RRT which are critical for Tirzepatide purification.

[0062] Comparison of Tirzepatide purity profile from Acidic to Basic route and Basic to Acidic route.

[0063] Basic to Acidic route of purification showed better yield, and purity for Tirzepatide purification.

[0064] BRIEF DESCRIPTION OF THE FIGURES

[0065] In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:

[0066] Figure 1: Illustrates the HPLC pattern of crude Tirzepatide of Formula I.

[0067] Figure 2: Illustrates the HPLC pattern of Tirzepatide of Formula I after purification with 0.05% OSA.

[0068] Figure 3: Illustrates the HPLC pattern of Tirzepatide of Formula I after purification without usage of TBAP.

[0069] Figure 4: Illustrates the HPLC pattern of Tirzepatide of Formula I after purification with usage of 0.075% TBAP.

[0070] Figure 5: Illustrates the HPLC pattern of Tirzepatide of Formula I after purification from Acidic route followed by Basic route

[0071] Figure 6: Illustrates the HPLC pattern of Tirzepatide of Formula I after purification from Basic route followed by Acidic route

[0072] DETAILED DESCRIPTION OF THE INVENTION

[0073] The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also form the part of this specification and are intended to be included within the scope of it.

[0074] Synthesis of Tirzepatide:

[0075] Synthesis of Tirzepatide was performed by solid phase synthesis method. The method involving sequential coupling of 39 protected amino acids from Serine (39thamino acid) to Tyrosine (1stamino acid) followed by selective removal of protecting group on Lysine and coupling of side chain components either sequentially or as a fragment. The resultant protected Tirzepatide was then subjected to global deprotection and cleavage from the resin and isolated post purification. EXAMPLES

[0076] Process for purification of Tirzepatide:

[0077] Example 1: Involves 3 step purification using 2 basic pH RP HPLC’s followed by 1 acidic pH RP HPLC with ion pairing agents in each step.

[0078] Crude Tirzepatide, having ~ 40% purity and ~ 20% assay was suspended in Tris- HC1 buffer pH 9.0±0.2 with 0.075% Tetra butyl ammonium phosphate (TBAP). The crude solution was further mixed with Acetonitrile: IP A to ensure crude was completely dissolved. Finally, load was filtered and loaded onto a preequilibrated column for RP-HPLC-1 purification.

[0079] RP-HPLC-1 purification:

[0080] The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Basic Aqueous buffer pH 9.0±0.2 + TBAP; B: ACN: IP A). Fractions having purity ~ 75% was further taken for next stage purification.

[0081] RP-HPLC-2 purification:

[0082] Poolable fractions of RP-HPLC-1 were further purified by RP-HPLC-2 to resolve impurities and achieve higher product purity. The RP-HPLC 1 fractions was diluted using aqueous buffer, filtered, and loaded onto column for RP- HPLC-2 purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Basic Aqueous buffer pH 9.0±0.2 + TBAP; B: ACN: IP A). Fractions having purity >90% was further taken for next stage purification.

[0083] RP-HPLC-3 purification:

[0084] Poolable fractions of RP-HPLC-2 was further purified by RP-HPLC-3 to resolve impurities and achieve final targeted purity. The RP-HPLC 2 fractions was diluted using aqueous buffer, filtered, and loaded onto column for RP-HPLC-3 purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Acidic Aqueous buffer + Octane sulphonic acid (OSA) pH 2.0±0.2; B: ACN). Fractions having purity >98.5% was further taken for next stage purification.

[0085] Further product isolation from poolable fractions was done via pl precipitation by adjusting the pH of pooled fractions to pH 3.8±0.2; wherein Tirzepatide falls out from solution in precipitated form. This precipitated suspension was then centrifuged to isolate product in pellet form. Furthermore, pellet was resuspended in dilute NaOH, dissolved completely using sodium hydroxide solution and freeze dried in lyophilizer to obtain purified Tirzepatide with purity >99.0%. Lyophilized product was amorphous white hygroscopic solid stored at -20±5°C. The summary of stage wise purity of Tirzepatide is as shown below. The overall yield of the purified Tirzepatide was ~ 50%.

[0086] Example 2: Involves 3 step purification using 2 basic pH RP HPLC’s followed by 1 acidic pH RP HPLC with ion pairing agents in steps 2 & 3 only.

[0087] Crude Tirzepatide, having ~ 40% purity and ~ 20% assay was suspended in Tris- HC1 buffer pH 9.0±0.2. The crude solution was further mixed with Acetonitrile: IPA to ensure crude was completely dissolved. Finally, load was filtered and loaded onto a pre-equilibrated column for RP-HPLC-1 purification.

[0088] RP-HPLC-1 purification:

[0089] The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Basic Aqueous buffer pH 9.0±0.2; B: ACN: IP A). Fractions having purity ~ 70% was further taken for next stage purification.

[0090] RP-HPLC-2 purification:

[0091] Poolable fractions of RP-HPLC-1 were further purified by RP-HPLC-2 to resolve impurities and achieve higher product purity. The RP-HPLC 1 fractions was diluted using Basic aqueous buffer, filtered, and loaded onto column for RP- HPLC-2 purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Basic Aqueous buffer pH 9.0±0.2 +TBAP; B: ACN: IP A). Fractions having purity >90% was further taken for next stage purification.

[0092] RP-HPLC-3 purification:

[0093] Poolable fractions of RP-HPLC-2 was further purified by RP-HPLC-3 to resolve impurities and achieve final targeted purity. The RP-HPLC 2 fractions was diluted using Acidic aqueous buffer, filtered, and loaded onto column for RP- HPLC-3 purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Acidic Aqueous buffer + OSA pH 2.0±0.2; B: ACN). Fractions having purity >98.5% was further taken for next stage purification.

[0094] The summary of stage wise purity of Tirzepatide is as shown below. The overall yield of the purified Tirzepatide was ~ 33%.

[0095] Example 3: Involves 2 step purification using basic pH RP HPLC followed by acidic pH RP HPLC with ion pairing agents in each step. Crude Tirzepatide, having ~ 40% purity and ~ 20% assay was suspended in Tris- HC1 buffer pH 9.0±0.2 with 0.075% Tetra butyl ammonium phosphate (TBAP). The crude solution was further mixed with Acetonitrile: IP A to ensure crude was completely dissolved. Finally, load was filtered and loaded onto a preequilibrated column for RP-HPLC-1 purification.

[0096] RP-HPLC-1 purification:

[0097] The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Basic Aqueous buffer pH 9.0±0.2+TBAP; B: ACN: IP A). Fractions having purity ~ 85% was further taken for next stage purification.

[0098] RP-HPLC-2 purification:

[0099] Poolable fractions of RP-HPLC-1 was further purified by RP-HPLC-2 to resolve impurities and achieve final targeted purity. The RP-HPLC 1 fractions was diluted using acidic aqueous buffer, filtered, and loaded onto column for RP- HPLC-2 purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: acidic Aqueous buffer + OSA pH 2.0±0.2; B: ACN). Fractions having purity >98.5% was further taken for next stage purification.

[0100] The summary of stage wise purity of Tirzepatide is as shown below. The overall yield of the purified Tirzepatide was ~ 31%.

[0101] Example 4: Involves 2 step purification using basic pH RP HPLC followed by acidic pH RP HPLC with no ion pairing agent in step-2. Crude Tirzepatide, having ~ 40% purity and ~ 20% assay was suspended in Tris- HC1 buffer pH 9.0±0.2with 0.075% Tetra butyl ammonium phosphate (TBAP). The crude solution was further mixed with Acetonitrile: IP A to ensure crude was completely dissolved. Finally, load was filtered and loaded onto a preequilibrated column for RP-HPLC-1 purification.

[0102] RP-HPLC-1 purification:

[0103] The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Basic Aqueous buffer pH 9.0±0.2+TBAP; B: ACN: IP A). Fractions having purity ~ 90% was further taken for next stage purification.

[0104] RP-HPLC-2 purification:

[0105] Poolable fractions of RP-HPLC-1 was further purified by RP-HPLC-2 to resolve impurities and achieve final targeted purity. The RP-HPLC 1 fractions was diluted using acidic aqueous buffer, filtered, and loaded onto column for RP- HPLC-2 purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Acidic Aqueous buffer pH 2.0±0.2; B: ACN). Fractions having purity >98.0% was further taken for next stage purification.

[0106] The summary of stage wise purity of Tirzepatide is as shown below. The overall yield of the purified Tirzepatide was ~ 44%.

[0107] Example 5: Involves 2 step purification using acidic pH RP HPLC followed by basic pH RP HPLC with ion pairing agents in each step Crude Tirzepatide, having ~ 40% purity and ~ 20% assay was suspended in acidic aqueous buffer pH 2.0±0.2. The crude solution was further mixed with Acetonitrile: IPA to ensure crude was completely dissolved. Finally, load was filtered and loaded onto a pre-equilibrated column for RP-HPLC-1 purification.

[0108] RP-HPLC-1 purification:

[0109] The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: .Acidic Aqueous buffer +OSA pH 2.0±0.2; B: ACN: IPA). Fractions having purity ~ 90% was further taken for next stage purification.

[0110] RP-HPLC-2 purification:

[0111] Poolable fractions of RP-HPLC-1 was further purified by RP-HPLC-2 to resolve impurities and achieve final targeted purity. The RP-HPLC 1 fractions was diluted using basic aqueous buffer, filtered, and loaded onto column for RP- HPLC-2 purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Basic Aqueous buffer pH 9.0±0.2+TBAP; B: ACN: IPA). Fractions having purity >98.0% was further taken for next stage purification.

[0112] Further product isolation from poolable fractions was done via pl precipitation by adjusting the pH of pooled fractions to pH 3.8±0.2; wherein Tirzepatide falls out from solution in precipitated form. This precipitated suspension was then centrifuged to isolate product in pellet form. Furthermore, pellet was resuspended and dissolved completely using ammonium hydroxide solution and freeze dried in lyophilizer to obtain purified Tirzepatide with purity >98.50%. Lyophilized product was amorphous white hygroscopic solid stored at -20±5°C. The summary of stage wise purity of Tirzepatide is as shown below. The overall yield of the purified Tirzepatide was ~ 37%.

[0113] Example 6: Involves 2 step purification using basic pH RP HPLC followed by acidic pH RP HPLC with ion pairing agents in each step - 2ndRP-HPLC would involve HAS as ion-pairing agent.

[0114] Crude Tirzepatide, having ~ 50% purity and ~ 20% assay was suspended in Tris- HC1 buffer pH 9.0±0.2 with 0.075% Tetra butyl ammonium phosphate (TBAP). The crude solution was further mixed with Acetonitrile: IP A to ensure crude was completely dissolved. Finally, load was filtered and loaded onto a preequilibrated column for RP-HPLC- 1 purification.

[0115] RP-HPLC- 1 purification:

[0116] The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: basic Aqueous buffer pH 9.0±0.2 +TBAP; B: ACN: IP A). Fractions having purity ~ 90% was further taken for next stage purification.

[0117] RP-HPLC-2 purification:

[0118] Poolable fractions of RP-HPLC- 1 was further purified by RP-HPLC-2 to resolve impurities and achieve final targeted purity. The RP-HPLC 1 fractions was diluted using acidic aqueous buffer, filtered, and loaded onto column for RP- HPLC-2 purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Acidic Aqueous buffer +Hexane sulphonic acid (HSA) pH 2.0±0.2; B: ACN). Fractions having purity >98.0% was further taken for next stage purification.

[0119] The summary of stage wise purity of Tirzepatide is as shown below. The overall yield of the purified Tirzepatide was ~ 48%.

[0120] Example 7: Screening of cationic ion pairing agent to purify the closely associated impurities.

[0121] Crude Tirzepatide, having ~ 40% purity and ~ 20% assay was suspended in Tris- HC1 buffer pH 9.0±0.2 along with a cation ion pairing agent. The crude solution was further mixed with Acetonitrile: IPA to ensure crude was completely dissolved. Finally, load was filtered and loaded onto a pre-equilibrated column for RP-HPLC-purification. The column was packed with C-8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Basic Aqueous buffer pH 9.0±0.2 +cation pairing agents; B: ACN: IPA). Different cationic ion pairing agents were screened to study the effect on purity. Below table provides the purity achieved using various cationic ion pairing agents in each RP-HPLC step.

[0122] The above example provided that, the use of TBAP resulted in higher purity of Tirzepatide.

[0123] Various experiments were conducted to evaluate the optimum concentration of TBAP required for obtaining high purity and maximum yield. Experiment outcome as below.

[0124] TBAP helps in resolution of closely eluting impurities such as 0.98 & 1.02 RRT which are critical for Tirzepatide purification Example 8: Screening of anionic ion pairing agent to purify the closely associated impurities.

[0125] Crude Tirzepatide, having ~ 40% purity and ~ 20% assay was suspended in acidic aqueous buffer pH 2.0±0.2 along with a anion pairing agent. The crude solution was further mixed with Acetonitrile optionally along with IPA to ensure crude was completely dissolved. Finally, load was filtered and loaded onto a preequilibrated column for RP-HPLC-purification. The column was packed with C- 8 substituted silica resin (10-13p). The bound product was eluted using a linear gradient of (A: Acidic Aqueous buffer pH 2.0±0.2 + anion pairing agents; B: ACN: IPA). Different anionic ion pairing agents were screened to study the effect on purity. Below table provides the purity achieved using various anionic ion pairing agents in each RP-HPLC step.

[0126] OSA helps in resolution of closely eluting impurities such as 0.97 & 1.02 RRT which are critical for Tirzepatide purification. HSA was also evaluated as an alternative to OSA but impurities resolution was not there and achieved only 92.5% Tirzepatide purity as compared to 96.28% purity with OSA.

Claims

CLAIMS1. A method of purifying crude Tirzepatide by RP-HPLC method using cationic ion pairing agent.

2. The method of purifying crude Tirzepatide according to claim 1, by RP- HPLC method using a cationic ion pairing agent to resolve closely associated impurities at RRT’s 0.90, 0.92, 0.96, 0.97, 0.98, 1.02, 1.05 & 1.06.

3. A method for purifying crude Tirzepatide, the method comprising of: a) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a cationic ion pairing agent along with the buffer b) subjecting the Tirzepatide from step a) to a second RP-HPLC purification using a anionic ion pairing agent along with the buffer c) Isolating the purified Tirzepatide.Wherein, optionally steps a or b or steps a & b are repeated, and steps a & b are performed in reverse order.

4. The method of purifying crude Tirzepatide according to claim 3, the method comprising of: a) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a cationic ion pairing agent along with the buffer with basic pH b) subj ecting the Tirzepatide from step a) to a second RP-HPLC purification using a anionic ion pairing agent along with the buffer with acidic pH c) Isolating the purified Tirzepatide.Wherein, optionally steps a & b are repeated.

5. The method of purifying crude Tirzepatide according to claim 3, the method comprising of: a) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a cationic ion pairing agent along with the buffer with basic pHb) subj ecting the Tirzepatide from step a) to a second RP-HPLC purification using a cationic ion pairing agent along with the buffer with basic pH c) subjecting the Tirzepatide from step b) to a third RP-HPLC purification using a anionic ion pairing agent along with the buffer with acidic pH d) Isolating the purified Tirzepatide.

6. The method of purifying crude Tirzepatide according to claim 4, to resolve closely associated impurities at RRT’s 0.90, 0.92, 0.96, 0.97, 0.98, 1.02, 1.05 & 1.06.

7. A method for purifying crude Tirzepatide, the method comprising of: a) dissolving the crude Tirzepatide using a cationic ion pairing agent along with the buffer b) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a cationic ion pairing agent along with the buffer c) subjecting the Tirzepatide from step a) to a second RP-HPLC purification using a anionic ion pairing agent along with the buffer d) Isolating the purified Tirzepatide.Wherein, optionally steps a & b are repeated.

8. A method for purifying crude Tirzepatide, the method comprising of: a) dissolving the crude Tirzepatide using a anionic ion pairing agent along with the buffer b) subjecting the solution of crude Tirzepatide to a first RP-HPLC purification using a anionic ion pairing agent along with the buffer c) subjecting the Tirzepatide from step a) to a second RP-HPLC purification using a cationic ion pairing agent along with the buffer d) Isolating the purified Tirzepatide.Wherein, optionally steps a & b are repeated.

9. The method of any of the above claims, wherein the cationic pairing agent is selected from a quaternary ammonium compound and anionic pairing agent is selected from alkane sulfonic acid.

10. The method of claim 8, wherein the alkane sulfonic acid is selected from the group consisting of octane sulfonic acid and hexane sulfonic acid.

11. The method of claim 8, wherein the quaternary ammonium compound is selected from the group consisting of tetra butyl ammonium phosphate, tetra butyl ammonium chloride, decyl trimethylammonium, and tetra butyl ammonium hydroxide.

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